Amebiasis is a parasitic disease provoked by the protozoan Entamoeba histolytica. It affects mainly the inhabitants of developing countries. Under appropriate conditions, which are not well known, trophozoites differentiate into an ineffective form or cyst, which is present in excrements and by this route can infect a new host by oral ingestion of food and water or person to person transmission. Most of the people infected with Entamoeba histolytica are asymptomatic, but in 10% of the people with amebiasis, the protozoan produces sickness when it invades the intestinal mucosa producing amoebic colitis or more dangerous damage when the protozoan is extraintestinal and there is a dissemination of the protozoan to the liver, provoking an amoebic liver abscess. In the cases in which there is a perforation of the liver or the intestine, it can provoke pleural damage, pericarditis, peritonitis and even death. Amebiasis occupies the sixth place among the most frequent causes of death in Mexico. In Mexico as in Venezuela, 2% to 15% of the cases of children with diarrhea who have been hospitalized, have infections associated with an Entamoeba histolytica.
To conduct correct epidemiological studies, it will require the development of diagnostic methods which are sensitive and specific. The coproparasitoscopic diagnosis of Entamoeba histolytica is especially difficult, because it requires highly skilled workers to prevent false interpretations. The serologic diagnosis is not effective because the existing tests are not sensitive enough, especially when they are used in highly endemic zones. To obtain useful diagnostic tests, it is necessary to know the amoebic molecules that are actively involved in the cases of invasive amebiasis and to utilize these molecules to design effective diagnostic tests. Once the role of these molecules is known, studies can be performed to determine their involvement in the immune protection mechanisms generated against amoebas and the possible implementation of vaccines.
A major impediment to achieve this goal is the highly elevated enzymatic activity of the proteases present in the amoebic extracts (see McLaughlin et al., Canadian Journal of Microbiology, 23: 420-425 (1977), and Perez-Monfort, et al., Molecular and Biochemical Parasitology, 26: 87-98 (1987)). The proteases degrade proteins in the amoebic extracts by producing degradation products and making it impossible or at least very difficult, to standardize the methods of analysis of the antigenicity of these proteins. To prevent this enzymatic activity, enzymatic inhibitors have been used; however, because these inhibitors are not completely effective protein degradation continues. Generally, amebic extracts containing enzymatic inhibitors only permit working with extracts for relatively short periods of time and they do not provide the opportunity to store the same samples for later tests. A later study by Arguello-Garcia et al., Arch. Invest. Med. (Mex.) 21: 3-9 (Supl. 1) (1990) reported evaluating E. histolytica antigens utilizing different protein extraction methods. The highest yield of proteins from E. histolytica cell lysates was obtained by homogenizing trophozoites in the presence of 10 mM p-hydroxymercuribenzoate (pHMB) and by lysis with Triton X-100 and a mixture of protease inhibitors. Frozen extracts were found to be stable for a period of two or three months. However, these extracts were not subjected to repeated freezing and thawing (personal communication). The authors found that different methods for preparing amebic extracts resulted in differences in protein yields as well as antigenic composition as observed by electrophoretic patterns and that there was a need to select a standardized procedure for preparing amebic extracts for use in serological assays.
The present invention utilizes a method for preparing amebic extracts based upon the method described in Said-Fernandez et al., Zeitschrift fur Parasitenkunde, 56: 219-225 (1978), which removes lipids from amebic extracts. This method disloses that the electrophoretic analysis of total proteins from trophozoites of four different strains of the histolytica group of Entamoeba show a striking similarity between the two E. histolytica strains studied. The authors state that protein electrophoretic analysis can be used as a taxonomic criterion after confirming its use in a sufficiently large number of strains.
The present inventor has determined that amoebic antigens prepared by this method exhibit very low levels of proteolytic enzymatic activity which does not cause protein any extensive and interfering protein degradation. Amebic extracts prepared by this method are stable for at least six months without any enzymatic degradation. Therefore, antigens made by this method for use in diagnostic tests, such as ELISA or IHA, are stable for longer periods of time than extracts containing protease inhibitors. The longer shelf-life provides advantages for a commercial diagnostic product amebic antigens prepared according to this method.
The present inventor has recognized that amebic extracts produced according to this method could be utilized as a stable antigen in a diagnostic test, such as an ELISA or IRA, without utilizing protease inhibitors to prevent the degradation of the amebic extract which is the standard treatment in the prior art. Therefore, the present invention is directed to a diagnostic test and method for detecting the presence of antibodies to E. histolytica in a patient sample.
Further, the present inventor has recognized that amebic extracts produced according to this method could also be utilized as a starting material to produce and characterize specific E. histolytica proteins associated with different clinical manifestations of amebic disease; e.g., amebic liver abscesses (ALA) and intestinal amebiasis (IA). These specific proteins may be utilized as stable antigens in diagnostic tests, such as ELISA and IHA for detecting the presence of antibodies to E. histolytica in patient samples or further utilized in the production of a vaccine against E. histolytica.